Compositions and methods related to tumor activated antibodies targeting EGFR and effector cell antigens

Abstract

Provided herein are multispecific antibodies for redirecting T cells to cancers, that rely on binding of one antigen interacting portion of the antibody to a tumor-associated antigen or marker, such as epidermal growth factor receptor (EGFR), while a second antigen interacting portion can bind to an effector cell antigen on a T cell, such as CD3, pharmaceutical compositions thereof, as well as nucleic acids, and methods for making and discovering the same.

What is claimed is: 
     
       1. An isolated polypeptide complex comprising an anti-epidermal growth factor receptor (EGFR) binding domain that is linked to a peptide that impairs binding of the anti-EGFR binding domain to EGFR, wherein the peptide comprises an amino acid sequence according to the formula:
 X 1 —C—X 2 —X 3 —X 4 —X 5 -D-X 6 -A-X 7 —P—X 8 —C—X 9 , (SEQ ID NO: 841) wherein 
 X 1  is selected from the group consisting of: P and L; 
 X 2  is selected from the group consisting of: R, L, T, A, N, I, V, S, H, and P; 
 X 3  is selected from the group consisting of: S, P, F, and Y; 
 X 4  is selected from the group consisting of: H, L, Q, P, R, F, and N; 
 X 5  is selected from the group consisting of: I, F, Y, H, N, T, S, D, A, L, and V; 
 X 6  is selected from the group consisting of: T, P, N, L, I, V, S, D, H, A, and Y; 
 X 7  is selected from the group consisting of: K and Y; 
 X 8  is selected from the group consisting of: I, P, L, and M; and 
 X 9  is selected from the group consisting of: A, V, I, T, L, S, D, F, V, and H, 
 wherein the anti-EGFR binding domain comprises heavy chain complementarity determining regions HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19, and HC-CDR3: SEQ ID NO: 20; and the anti-EGFR binding domain comprises light chain complementarity determining regions: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 16, and LC-CDR3: SEQ ID NO: 17. 
 
     
     
       2. The isolated polypeptide complex of  claim 1 , wherein
 X 1  is selected from the group consisting of: P and L; 
 X 2  is selected from the group consisting of: R, L, T, A, and N; 
 X 3  is selected from the group consisting of: S, P, and F; 
 X 4  is selected from the group consisting of: H, L, Q, and P; 
 X 5  is selected from the group consisting of: I, F, Y, H, N, and T; 
 X 6  is selected from the group consisting of: T, P, N, L, I, and V; 
 X 7  is selected from the group consisting of: K; 
 X 8  is selected from the group consisting of: I; and 
 X 9  is selected from the group consisting of: A, V, I, T, L, and S. 
 
     
     
       3. The isolated polypeptide complex of  claim 2 , wherein
 X 1  is selected from the group consisting of: P; 
 X 2  is selected from the group consisting of: R, L, and T; 
 X 3  is selected from the group consisting of: S; 
 X 4  is selected from the group consisting of: H, L, Q, and P; 
 X 5  is selected from the group consisting of: I, F, Y, and T; 
 X 6  is selected from the group consisting of: T, P, N, and V; 
 X 7  is selected from the group consisting of: K; 
 X 8  is selected from the group consisting of: I; and 
 X 9  is selected from the group consisting of: A, V, and I. 
 
     
     
       4. The isolated polypeptide complex of  claim 1 , wherein the peptide comprises the amino acid sequence according to any one of SEQ ID NOs: 99-118. 
     
     
       5. The isolated polypeptide complex of  claim 1 , wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 115. 
     
     
       6. The isolated polypeptide complex of  claim 1 , wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 116. 
     
     
       7. The isolated polypeptide complex of  claim 1 , wherein the peptide comprises the amino acid sequence according to any one of SEQ ID NOs: 26, 87, 90, 92, or 96. 
     
     
       8. The isolated polypeptide complex of  claim 1 , wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 26. 
     
     
       9. The isolated polypeptide complex of  claim 1 , wherein the anti-EGFR binding domain comprises an antibody or an antibody fragment. 
     
     
       10. The isolated polypeptide complex of  claim 9 , wherein the antibody or antibody fragment comprises a single chain variable fragment, a single domain antibody, a Fab, or a Fab′. 
     
     
       11. The isolated polypeptide complex of  claim 10 , wherein the antibody or antibody fragment comprises the Fab. 
     
     
       12. The isolated polypeptide complex of  claim 11 , wherein the Fab comprises a light chain polypeptide having the amino acid sequence of SEQ ID NO:21 and a heavy chain polypeptide having the amino acid sequence of SEQ ID NO:24. 
     
     
       13. The isolated polypeptide complex of  claim 11 , wherein the Fab comprises a light chain polypeptide having the amino acid sequence of SEQ ID NO:21 and a heavy chain polypeptide having the amino acid sequence of SEQ ID NO:23. 
     
     
       14. The isolated polypeptide complex of  claim 11 , wherein the Fab comprises a light chain polypeptide having the amino acid sequence of SEQ ID NO:22 and a heavy chain polypeptide having the amino acid sequence of SEQ ID NO:23. 
     
     
       15. The isolated polypeptide complex of  claim 11 , wherein the Fab comprises a light chain polypeptide having the amino acid sequence of SEQ ID NO:22 and a heavy chain polypeptide having the amino acid sequence of SEQ ID NO:24. 
     
     
       16. The isolated polypeptide complex of  claim 1 , wherein the anti-EGFR binding domain is linked to the peptide through a linking moiety. 
     
     
       17. The isolated polypeptide complex of  claim 16 , wherein the linking moiety is a substrate for a tumor specific protease. 
     
     
       18. The isolated polypeptide complex of  claim 17 , wherein the tumor specific protease is selected from the group consisting of a matrix metalloprotease (MMP), a serine protease, a cysteine protease, a threonine protease, and an aspartic protease. 
     
     
       19. The isolated polypeptide complex of  claim 16 , wherein the linking moiety is bound to an N-terminus of the anti-EGFR binding domain. 
     
     
       20. The isolated polypeptide complex of  claim 16 , wherein the linking moiety is bound to a C-terminus of the anti-EGFR binding domain. 
     
     
       21. The isolated polypeptide complex of  claim 16 , wherein the linking moiety is a peptide sequence having at least 5 to no more than 50 amino acids.